Ischemia indicates a local anemia which is caused by clogging or narrowing of blood vessels. Every part of human body needs to be supplied enough oxygen and nutrition through blood vessels to be functioning normally. The heart also needs to be provided with oxygen and nutrition through the blood vessel called the coronary artery. With insufficient blood flow, metabolites are accumulated in the myocardium, leading to hypoxia with causing functional disorder. This case is called myocardial ischemia and the heart failure resulted from the myocardial ischemia is called ischemic heart disease, which is largely represented by angina pectoris and myocardial infraction. In general, the incidence of such heart disease is higher in male than in female, as being older and in those having risk factors. Once ischemia is developed, blood vessels are apt to be blocked, resulting in the said diseases, for example the interruption of brain vessels results in ischemic heart disease. Once the blood circulation is interrupted by ischemia, terminal regions are going to be in trouble too, that is terminal tissues are necrotized to cause ischemic limb injury. As explained above, ischemia is a trigger for various diseases, so it is an urgent request to develop a therapeutic agent targeting ischemia.
HIF-1 (hypoxia-inducible factor-1) is a key regulator of hypoxic adaptation that functions by activating the transcription of several genes involved in angiogenesis, erythropoiesis, and glycolysis (Masson and Ratcliffe, J Cell Sci 116:3041-3049, 2003; Seagroves et al, Mol Cell Biol 21:3436-3444, 2001). It consists of two subunits; HIF-1α is rapidly degraded under normoxic condition by the ubiquitin-proteasome system, whereas HIF-1β is stable (Huang et al., Proc Natl Acad Sci USA 95:7978-7992, 1998; Kallio et al, J Biol Chem 274:6519-6525, 1999). Under normoxic conditions the proline-564 and/or 402 residues of HIF-1α are hydroxylated by PHDs (HIF-1α-specific prolyl-4-hydroxylases) and thus inhibited, which needs O2, 2-oxoglutarate, vitamin C and Fe2+ (Bruikc and McKnight, Science 294:1337-1340, 2001; Epstein et al., Cell 107:43-54, 2001; Ivan et al., Science 292:464-468, 2001; Jaakkola et al., Science 292:468-472, 2001; Masson et al., EMBO J 20:5197-5206, 2001). PHD1 (HPH3, EGLN2), PHD2 (HPH2, EGLN1) and PHD3 (HPH1, EGLN3) are the PHD family found in mammalian cells (Huang et al., J Biol Chem 277:39792-39800, 2002; Talyer, Gene 275:125-132, 2001). The hydroxylated prolines interact with VHL (von Hippel-Lindau) protein, a component of E3 ubiquitin ligase, and the HIF-1α is ubiquitinated by the VCB E3 ubiquitin-ligase complex (VHL protein, Elongin B, Elongin C, Cul2 and Rbx1) (Iwai et al., Proc Natl Acad Sci USA 96:12436-12441, 1999; Kamura et al, Science 284:657-661, 1999). In hypoxic conditions, proline hydroxylation decreases and HIF-1α accumulates. The accumulated HIF-1α migrates to the nucleus to interact with HIF-1β in order to be activated as a transcription factor. HIF-1α/β hetero-complex binds to HRE (hypoxia-responsive elements) located in the promoter region of a target gene and to transcription co-factors such as p300/CBP and thus increases the expressions of target genes necessary for angiogenesis and intracellular oxygen supply (Nathali et al., Biochem Pharmacol. 68:971-980, 2004).
Oxygen molecules inhibit not only the stabilization of HIF-1α but also its transcription activity, since FIH-1 (factor inhibiting HIF-1α) catalyzes hydroxylation of the asparagine-803 residue of HIF-1α. Hydroxylation of the asparagine residue in the transactivation domain of HIF-1α prevents it from recruiting its coactivator CBP and thus induces the accumulation of non-functional HIF-1α. FIH-1 has been recently identified and followingly its activity and tertiary structure have also been identified (Lee et al., J. Biol. Chem. 278:7558-7563, 2003; Elkins et al., J. Biol. Chem. 278:1802-1806, 2003). Like PHD, FIH-1 also recruits Fe2+ as a cofactor and catalyzes hydroxylation of the asparagine-803 residue of HIF-1α using O2 and 2-oxoglutarate (Lando et al., Science 295:858-865, 2002). FIH-1 has at least 2-fold lower Km for 2-oxoglutarate and oxygen than PHD indicating that it is functioning even under inoperable oxygen pressure (Koivunen et al., J. Biol. Chem. 279:9899-9904, 2004).
Target genes of HIF-1α, EPO (erythropoietin) and VEGF (vascular endothelial growth factor) act as an important mediator of protective mechanism under hypoxic condition (Grimm et al., Nat Med 8:178-724, 2002: Calvillo et al., Proc Natl Acad Sci USA 100:4802-4806, 2003: Ferriero D. M., Epilepsia. 46:45-51, 2005; Simons, A., Ware, J. A., Nat Rev Drug Discov. 2:863-871, 2003). EPO and its receptor are expressed in the brain and thus involved in neuroprotection in relation to cerebral infarction associated brain damage (Digicayolioglu, M. et al., Proc Natl Acad Sci USA 92:3717-3720, 1995; Ehrenreich, H. et al., Metab Brain Dis. 19:195-206, 2004), that is they presumably protect neurons when cerebral infarction is developed in vivo (Sasaki et al., Proc Natl Acad Sci USA 95:4635-4640, 1998). VEGF has also been evaluated as a stable therapeutic agent for myocardial infarction (Yoon, Y. S. et al., Mol Cell Biochem. 264:1494-1504, 2004; Shah, P. B. and Losordo, D. W., Adv Genet. 54:339-361, 2005; Simons, M. and Ware J. A., Nat Rev Drug Discov. 2:863-871, 2003; Henry, T. D. et al., Circulation 107:1359-1365, 2003). VEGF has a protective activity to neuron and neuroglia damaged by angiogenesis (Rosenstein, J. M. and Krum, K, Exp Neurol 187:246-253, 2004). When VEGF level is decreased, amyotrophic lateral sclerosis is developed and perfusion and neuroprotection functions are reduced (Storkebaum, E., Lambrechts, D., Carmeliet, P., Bioessays 26:943-954, 2004).
Clioquinol (referred as “CQ” hereinafter) is involved in selective chelation of heavy metal ions such as Zn2+, Cu2+ and Ca2+ and regulates the effects of such metal ions on enzyme activity and protein formation. pKa values of CQ are as follows: Cu2+, 15.8; Zn2+, 12.5; Ca2+, 8.1; Mg2+, 8.6 (Agrawal, Y. K. et al., J Pharm Sci. 75:190-192, 1986). CQ is hydrophobic and able to cross the blood-brain barrier. CQ has been re-evaluated as the prototype metal-protein attenuating compound that reduces metalloprotein precipitation in Alzheimer's disease, Parkinson's disease and Huntington's diseases and oxidative stress thereby. CQ was extensively used as an antibiotic in the mid-1900s, but then withdrawn because it caused subacute myelo-optic neuropathy in Japan (Chery, R. A., Neuron 30:665-676, 2001; Kaur, D. et al., Neuron 37:899-909, 2003; Nguyen T. et al., Proc Natl Acad Sci USA 102:11840-11845, 2005). In a study of APP2576 transgenic mice which have an Alzheimer's disease-type neuropathy, CQ reduced both amyloid beta plaques and serum levels of amyloid beta, without systemic adverse effects (Doraiswamy, P. M. et al., Lancet Neorul 3:431-434, 2004). A recent phase II clinical trial in 36 patients with Alzheimer's disease showed that CQ slowed cognitive decline and decreased plasma amyloid beta concentrations (Ritchie C. W. et al., Arch Neurol 60:1685-1691, 2003).
CQ also causes apoptotic cell death in several human cancer cell lines. The addition of copper or zinc to the CQ treated cancer cell lines did not prevent cell death and rather increased the apoptotic cell death. CQ treated cells were fluorescence-labeled to detect the level of zinc. And from the result was confirmed that CQ induced cell death by activating zinc ionophore (Ding, W. O., et al., Cancer Res. 65:3389-3395, 2005).
TPEN (tetrakis-(2-pyridylmethyl)ethanediamine: C26H28N6) is also a metal chelator like CQ, which is involved in the selective chelation of heavy metal ions such as Zn2+, Cu2+ and Fe2+ and regulates the effects of such ions on enzyme activity and protein formation. pKa values of TPEN are as follows: Cu2+, 20.2; Zn2+, 15.4; Fe3+, 14.4; Ca2+, 3 (Chemy R. A. et al., J Biol Chem., 274:23223-23228, 1999).